CA2101557A1 - Pliable biological graft materials and their methods of manufacture - Google Patents

Abstract

PLIABLE BIOLOGICAL VASCULAR GRAFTS AND
THEIR METHODS OF MANUFACTURE

Abstract Preparing chemically cross-linked collagenous biological graft material (10a) by preparation processes which include the step of altering the locations and/or orientations of chemical cross-linkages formed during the collagen cross-linking process. Embodiments of the method include various processes whereby physical force, stress or movement is applied to alter the relative positioning of the collagen fibers within the graft materials (10a) during at least the initial period of exposure to the collagen cross-linking reagent.

Description

35~ ~
~1--P~IAB~ BIO~OG~CA~ MA~RIa~ A~D
~ ~OD~ ~ ~A~ T~
Fi~1~ o~ the I~v~n~io~ ~
This invention relates generally to ~he preparation .
of biological tissue gra~ts and more improved method~ of L
preparing che~ically cross-linked collagenous gra~t materials having improved flexibility and pliability.
~a~kgroun~ o~ th~ ~nventlon Natural tissues obtalned from mammalian sources ha~e been used for allogenic and xenogeneic ~rafting for many years. For xample, certain cardiovas~ular tiæsues ~e,g.
segments of blood vessel, h~art valves) have been harvested from human or other mammalian sources and subsequently surgically implanted in the human body.
Many of the biological tissues which one desires to harvest and prepare ~or subsequent surgical implantation contain substantial amounts of connective tissue. The function of the connective tiæsue i5 to provide a supportive frame work within which other functional cell types (e.g. muscle fibers) are disposed. Connective ti~sues are largely formed of insoluble proteins ~oll~gen and el~sti~. Collagen and elastin exist in the form of insoluble fibers. Such insoluble fibrils are arranged . .
within a continuous matrix called the grou~d sub~ta~ae.
The flexibility and other characteristics of the : connective tissue depend largely on the proportion~ of collagen and elastin con~ained within suc~ tiæsue, and .

~LaL~7 - z -the structur~ and configura~ion of ~he ~ollag~/elastic ~iber n~twork thereo~.
As illustrated in Figures 5a-5c herein, each collagen molecule consists of thr~e polypeptide chains intertwined in a coiled helical conforma~ion, The individual amino acid constituents of each polyp~ptide chain are connected, by hydrogen bonds, to individual amino acids o~ an adjacent pol~peptide chain, thereby holding the individual polypeptide chains in the triple helical conformation shown in Figure 5c.
The current methods o~ preserving and preparing collagenous, biological tissues ~or subsequent surgical implantation includa the step of "fixing" the collagen network by exposing the tissue to one or more chemical compounds capable of cross-linking the collagen molecule~
within the tissue. Both intermolecular (FIG. 5b) and intramolecular (FIG. 5c) cross-linkages may be formed by the currently known ~ixative compounds.
Chemical compounds which are known to cross link collagen include ~ormaldehyde, glutaraldehyde, dialdehyde starch, hexamethylene diisocyanate and certain polyepoxy compounds including glycol diglycidyl ether, polyol polyglycidyl ether ~nd dicarboxylic acid diglycidylester.

Three (3~ specific water soluble polyepoxy compounds which may be u~ied as collagerl cross-liT~ing agents ar~3 shown below:

1. Difunctional_epoxy Ethylene glycol digly~idyl ekher ~molecular weight - 270) CH2-CH-~H2-O-(CH2~C~2~C)~)n-c~2-cH-c~2 (wherein: n=1) (I)enacoltm Ex-810; Nagase Chemicals, Ltd., Osaka, Japan~

2. Trlfunctional epoxy Glycerol Triglycidyl ether .
(molecular weight - 435) CH2--0--CH2-ClH--CH2 CH--O--CH2 CH--CH2 , O

(Denacol~m Ex-313; Nagase Chemicals, Ltd., Osaka, Japan) . . . _ _ 3.Tetra~unctional e~oxy~
40Sorbitol tetraglycidyl ether (molecular weight = 680) OH OH
45CH2-CH-CH2-0~CH2-CH-CH-CH-CH-CH2-0--~H2-CH-CH2 CH2-CH-CH2-0 o-CH2-CH-~ H2 50(Denacoltm Ex 612; Nagase Chemicals, Ltd., O~aka, Japan) _4 210~``3~ ~

In general, the low molecular weight chemical fixatives, such a~ glutaraldehyde, are relativ~ly fast acting. On the other hand, high molecular weight ~ixatives, such as polyepoxy compounds, are relatively slow actinq. Thus, the exposure time required to effect adequat~ cross-linking of a collagenous graft dep~nds on the molecular weight and ~unctionality (i.e. the reactivity) of the cross-linking reagent being used.
Examples of biological graft materials and methods of preparation thereof are described in U. S. Pakent Nos.
2,900,644 (Rosenberg et al.), 3,927,422 (Sawyer), 3, 966, 401 (Hancock et al. ), 3, 974, 526 (Dardik et al.), 4,239,492 (Holman et al.), 4,466,139 (Ketharanathan et al.) and 4,806,595 (Noishiki et al.).
One drawback associated with the use of chemically cross-linked collagenous graft materials is that the cross-linking of the collagen molecules within the graft typically results in undesirable stiffness and a loss of pliability. Such sti~fening or loss of pliability may 20 render the biological graft difficult to suture and difficult to manipulate during surgical implantation. In particular, with respect to arterial vascular grafts, the unnatural stiffness of an implanted biological graft may result in poor hemodynamic performance of the graft, with 25 possible resultant clinical ~ailure thereof. See, Abbott W. M. and Cambria, R. P., Control Of Physical Characteristics - Elasticity and Compliance of Vascular Grafts; BIOLOGIC AND SYNTEIETIC VASCUI~R PROSTHESES, Stanley~ J. c. et al. ~ed) (Grune ~ stratton, 1982).
In Yiew o~ the unde~irable stiffness of chemically r cross-linked collagenous biological graft materials of 5 the prior art, there exists a need for the development of improved methods of preparing and preserving such collagenous biomaterials whereby the xesultank stif~ness and lack o~ pliability may be avoided or minimized.
The graft preparation methods o* the present lO invention are particularly applicabl~ to, though not limit~d to, the preparation of vascular grafts such as coronary artery bypass grafts. Indeed, blood vessels are known to contain relatively large amounts of collagen.
The quantity o~ collagen found in a particular blood 15 vessel appears to vary with the types of mechanical forces whioh affect that blood vessel under normal physiological conditions. In vessels which typically encounter low pressure (e.g. small veins~ the collagen content relative to the diameter of the vessel is low.
20 On the othar hand, in vessels which encounter high ; ~pressure (e.g. arteries) the collagen content relative to the diameter of the vessel is high. Indeed, collagen is known to con~titute approximately 20% of the dry weight of some large elastic arteries and perhaps up to 50% of 25 the dry weight o~ some smaller peripheral arteries.
Thus, in view of~ (a) the desirability o~ using preserved collagenous biological tissues (e.g. blood vessels) ~or allogenic or xenogeneic grafting and (b) th-:~ :
..

propensity for such collagenous tissues to becc:me s;ti~f when treated with the known collagen cross-linking reagents, it is d~sirable to deYelop new methods of preserving or fixing such collagenous graft materials so 5 as to improve the resultant pliability and flexibility there~of .
~u~marY ~ th~ I~v~tio~
The present invention overcomes some or all of th~
foregoing shortcomings o~ the prior art by providing an improved m~t~od for preparing collagenous biomaterial ~or subsequ~nt surgical impl~ntation into the human body. In general, the invention comprises a method of preparing chemically cros~-linked collagenouæ biological materials wherein the locations and/or orientations o~ the chemical 15 croqs-linkages formed within and/or between collagen molscules arë caused to be altered from those which would normally be formed if the graft material ware to be immersed in or otherwise exposed to chemical collagen cros~linking chemicals while in an unstressed relaxed state.
One means by which the locations and/or orientations of the collagen cross-linkages may be altered, in accordance with the present invention, is by physically changing the relative spacial and positional relationships of the individual collagen fibers and/or molecules within the tissue during at least a portion of the chemical cross-linking (i.e. chemical fixation) process. Such changes of spacial or positional , I
relationships of the collagen fibers may be accomplished by physically stre~sing, moving, relocating, vibrating or otherwise applying force to the graft material. Such changing of the relati~e spacial or positional S relationships o~ the collagen ~ibe~s a~dJor collagen molecules will result in modification of ~he locations and/or orientation of the chemical cross-linkage formed between and/or within collagen molecules as oppus~d to the locations and orientations of chemical cross-linkages which would normally have been formed i~ the chemical cross linking proce~s had been carried out without such physical movement~ stressing or physical relocation of the individual collagen fibers.
In accordance with the invention, the physical movement, stressing or relocation of the collaye fibers may ~e carried out by exerting physical force on the graft material while the graft material is immersed in a collagen cross-linking reagent. The exertion of force upon the graft material that may be carried out statically while the graft material remains exposed to the cross-linking reagent or may constitute a dynamic process whereby th~ exertion of force upon and/or movsment of the graft material is carried out intermittently and repeatedly during at least a portion of the time that the graft material remains exposed to the collagen cross-linking reagent.
Further in accordance with the invention, the network of collagen fibers within the graft material may 21&1;.3~j7 be compressed while the collagen ~ibers are exposed ~o a collagen cross-linking reagent. In cases where ~he graft material is of elongate tubular con~iguration (e.g. a blood vessel~, the elongate graft may be physically compressed along its longitudinal axis and subsequently held in such longitudinally compressed state while bein~

immersed in a collagen cross-linking reagent, Further in accordance with the invention, the network of collagen fibers within the graft material may be alternatsly (a) stretched and ~b) relaxed during at least a part of the time that the collagen fibers are exposed to a collagen cross-linking reagent. In cases where the gra~t material is of elongate tubular con~iguration (e.g. a blood vessel~, the alternate stretching and relaxing may constitute lon~ltudinal stretching/relaxing along the longitudinal axis of the graft or radial stretching/ relaxing whereby the circumference of the tubular graft is alternately enlarged and relaxed.
In accordance with a still further aspect of the invention, the network of collagen fibers within the gra~t material may be alternately compressed and relaxed while the collagen ~ibers are exposed to a collagen cross-linking reagent. In cases where the biological graft material is of an elongate tubular configuration (e.g. a blood vessel), the intermittent compression/relaxation may comprise lon~itudinal compression/relaxation along the longitudinal axis of the ~ 5 graft or radial 5tretching/relaxing whereby the circumference of the ~ubular graft is alternately enlarged and relaxed.
The methods of preparing collagenous biological graft materials of the present invention may be utilized ln conjunction with any type of collagen cro5s-linking reagent. However, the time of application of the meth~ds of the present invention may vary~ depending on the reactivity of the particular cross-linking reaqent being used.
Various additional aspects, objects and advantag~s o~ the present invention will become apparent to those skilled in the art upon reading and understanding of the following detailed des~ription and the ac~ompanying drawings.
~rie~ DescriPti~ o~ the Dr~wi~g~
FIGS. la-ld is a schematic diagram illustrating a preferred method of preparing vascular gra~s by static longitudinal retraction;
FIGS. 2a-2a is a schematic diagram of a preferred method of preparing vascular grafts dynamic longitudinal stretching;
FIGS. 3a-3b i5 a diagram of a method and apparatus : for preparing pliable vascular grafts by dynamic radial stretching;
FIG. 4 is a diagram of a preferred method and apparatus for preparing pliable vascular grafts by dynamic radial contraction.

t -10 ~ .
FIG. 5a is an illu~tration o~ ~ typical cvllag~n fibril comprising five individual collagen molecules.
FIG. sb is a schematic illustration of segment b-b~
of Figure 5a, showing the positioning of intermolecular cross-linkages bQtween collag~n molecules.
FIG. 5c is an enlargad schematic illustration of L
segment c-c' ~f Figure 5b, showing the positioning of intramolecular cross-linkages between the polypeptide chains of the collagen molecule.
Detailed ~eV~ri~tio~ o~ the Pre~err~d ~boai~t The ~ollowing detailed descriptions and th~ examples set forth therein are pro~ided for the purpose of describing and illustrating prssen~ly pr~ferred embodiments o~ the invention. ~he following detailed description and examples of the presently preferred embodiments are not intended to limit the scope of the claims in any way.
A. ~ Firs~ Embodiment:
Statlc Betraction or comPression of the Colla~enous NQtwork durin~ Fixatio~
One embodiment of this invention compri9es placing and holding the collagenous graft material in a co~pressed ~i.e. retracted) state during exposure to one ; 25 or more collagen cross-linking agents.
In accordance with this embodiment of the invention, as illustrated in Figures la-ld, the collagenous graft material te.g. a segment of blood vessel) may be disposed on the surface o~ a tube, rod, mandrel, plate, jig or other form member. Preferably, the surface of the form membar is ~mooth and compatible with the configuration of the graft. The graft is then caused to remain in such compressed or retracted state by attachment of a holding r apparatus 14 such as clips~ clamps, sutures or tie wraps.
The graft iS caused to remain in such compressed or retracted state during at least the initial phase o~ the collagen cross-linking reaction as may be accomplished by immersing the retracted graft material in a pool of collagen cross-linking fixative solution.
The amount of compression or retraction applied Will vary, depending ~n the type of ~ollagenous tissue being treated. In vascular applications~ it is preferable that an elongate segment of blood vessel be longitudinally shortened by an amount equal to about 1% to 50% of its original relaxed length and most preferably 5% to 30~ of its original relaxed length.
Example 1:

Preparatio~ o~ a Pliablo_Coronary ~rtery Bypass Graft Un~r 8tat~o Lon~itu~in~l Retra~tion or Compresqion A segment of bovine coronary artery lOa is removed ~rom a donor animal and cut to a desired length (hereinafter referred to as the "relaxed length"). After the harvested segment of artery lOa has been thoroughly cleaned with sterile saline solution, any excess surrounding connective tissue is trimmed away using standard surgical instruments. Additionally, any unwanted bra~ches of the artery are liyated through the us~ of standard surgical technique and surgical suture materials, After such cleaning, trimming and ligation procedures have been completed, the segment of artery lOa is slidably advanced onto the outer surface of a cylindrical rod or othQr cylindrical support member 12.
The cylindrical support member 12 pref~rably has a diameter which is equal to or slightly larger than the internal diameter of the artery.
lo ~fter the artery lOa has been disposed upon cylindrical support member 12, the segment of artery lOa is longitudinally compressed or retractsd to a d~sired "compressed length" and ligatures or clips 14 are placed around the ends of the segment of artery lOa ~o hold the segment of artery lOa in the longitudinally retracted or compressed state. (FIG. lb). While being maintained in such retracted or compressed state, the segment of artery ~Oa is immersed (FIG. lc) in a bath 16 of chemi~al ~ixative solution (FIG. lc). In this example the *ixative solution comprises 2% by volume aqueous solution of glycerol triglycidyl ether (MW 435) Denacoltm Ex-313;
Nagase Chemicals Ltd., Osaka, Japan). ~his fixative solution iB maintained at room temperature and buffered : to a Ph of approximately lO.O by addition of 0.1 N
carbonate-bicarbonate` buffer solution. Other Denacolt~

epoxy materials may also be used as described in U. S.
Patent No. 4~806,515. The entire disclosure o~ U.S.

Patent No. 4,806,515 is expressly incorporated herein by '7 ~13--refer~nc~ for the purpose of disclosing the chemical compositions, prop~rties and methods 9i~ applicat~ on of the sp~cific Denacoltm epoxy materials as fixatlves in this invention.
It is preferable that the segment of artery lOa remain in the 2% Denacol Ex-313 solutiQn at room temperature and ambient pressure for about 10~80 hours, and most pre~erably about 20-60 hours. In this particular example the segment of artery lOa remained immersed in the 2% Denacol Ex-313 fixative solution at room temperature and ambient pressure for a total of 66 consecutive hours.
After 66 hours of immersion in the fixative bath 16, the segment of artery lOa was removed from the bath 16, ligatures 14 were removed and the segment of artery lOa was slidably extracted from the outer surface of the cylindrical suppoxt member 12.

TAB~E 1, below, contains modulus of elasticity da~a ~or one (1) ~resh untreated segment of bovine coronary artery (control) and six (6) segments of ~ovine carotid artery treated in accordance with this example. As shown, the six (6) segments o~ artery were compressed or retracted to lengths which were 0%, 10%, 20%, 30%, 40%
and 50% than the respective original unretracted length, o~ each segment of artery.

14 ~ ~ 0 ~3m~1~RelaxedCompressed % ~ob~lr~
No.Lenath ~sm)Len~th (cm) Retraction Elasticity (p~i) 9.1 9~1 0 ~9~2 B 10~5 9.4 10 45.4 c s.o 7~2 20 25.9 D 10~7 7.9 30 23.5 lo E 11.5 6.9 40 22.9 F 1002 5.1 50 6.6 G(Control) (Fresh, unfixed Bovine Coronary Artery) 13.0 The modulus of elasticity of each of the six (6) treated samples and the one (1) untreated ~control) sample are shown in TABLE 1. The modulus of elasticity of a biomaterial reflects its strain change as a result o~ applied stress. The lower the modulus of elasticity, th~ softer and more pliable the material.
As shown in TABLE 1, the modulus of elasticity of the treated samples ~A through F) varied inversely with the percentage of longitudinal retraction applied, with the highest modulus of elasticity being observed in the sample traated under the leas~ longitudinal retraction (0%) and the lowest modulus of elasticity being observed in the sample treated under the greatest longitudinal retraction (50%).
Given the f~ct that the measured modulus of elasticity of the untreated control sample (Sample G) was 13.0 psi, these data indicate that a modulus of elasticity equivalent to that of fresh ~nfixed coronary artery will bq observed in segments of artery fixed under a longitudinal compression of between 40% to 50% and, r~ I
-~5-thus, in this ex~mple it would be preferable to ~mploy a longitudinal compression of 40~ to 50%.
Current laboratory data indicate~ that, although static longltudinal compression has the e~fect of ~:
improving pliability of the cross-linked graft, no such improvement in graft pliability is observed when sta~ic longikudinal extension or ~tretching is applied.
B. A Second Embodiment:
_y~amic Manipulation of the Coliagenous 10Gra~t Material Durina Fixation The second preferred embodiment of the present invention comprises repeatedly manipulating or moving I .
(e.g. stretching, contracting, twisting, flexing) the collagenous graft material to cau~e dynamic movement or flexing of collagen fibers within the graft, relative to one another, while at least a portion of the collagen ~
cross-linking reaction is occurring. Such repetitive D
movement of the graft material causes changes in the positional and spacial relations of the collagen molecules and/or collagen fibers within the graft, relativ~ to one another, while the intra- and/or intermolecular crosæ-linkages are being formed. As a result, the intra- and/or intermolecular cross-linkages are formed at different locations on the collagen molecules and/or collagen fiber~; than if the cross-linking reaction were to have bean carried out while the graft remained in a relaxed, unstressad state.
This repetitive manipulation of the collagenous graft has its greatest effect on resultant pliability of y~

the graft when carried out during ~he initial period o~
expo~ure to the collagen cro~-linking fixative agent.
This heightensd ef~ectiveness dur~ng the initial stage o~
the collagen cross-linking reaction i~ likely due to the 5 fact that the individual collagen fibers are only free to move or change position relative to one another before a substantial number of inter- or intramolecular cross-linkages have been form~d. If, however, a substanti~l num~er of inter- or intramolecular cross-linkages are ~ormed before said dynamic manipulation is begun, the presence of ~uch cross-linkages may prevent the collagen fibers ~rom freely moving relative to one another, thereby preventing the dynamic manipulation from altering the locations and/or orientations of collagen cros.~
linkages ~rom those which would be formed if the gra~t were fixed without any such manipulation or movement thereo~. The actual time period during which the stretching-relaxation process is most e~fectively applied varies depending on the kinetics o~ the cross~linking ~o reaction. For example, when a fast acting cross-linking reagent such as glutaraldehyde is used, the collagen modulating effect is most effective during approximately the initial sixty (60) seconds of the fixation proces~.
: When a slow acting cross-linking raagent such as a polyepoxy compound is used, the collagen modula~ing effect is most effective during the first 0.1 to lOoO
hours of the fixation process, and preferably during the ~irst two (2) hours thereof.

One means of dynamically manipulating or moving the collagenous gra~t material during fixation is to subject t~e collagenous graft material to repetitive longitudinal stretching during at least a portion of the chemical cross-linking process.
In accordance with this aspect of the invention, the graft material may be fixed c~n the outer surface of a tubet rodl mandrel, platet jig or other ~upport member.
It is preferable that the support member have a smooth outer surface upon which the gra~t material may slide, thereky allowing the gra~t material to ke intermittently (a) stretched and (b) relaxed while remaining disposed on the surface of the support member.
one or more sides or ends of the yraft material is/are attached to the support member by way of an attachment member Such as a ligature or clip. The opposing sides and/or ends opposite the previously attached sides and/or ends of the gra~t material are then grasped by a movable clamp member(s) or other gripping apparatus. The graft material is then immersed in ~ixative solution and the movable clamp member(s~ is/are ~ moved away from the opposite sides and/or ends o~ the ; gra~t so as to stretch the graft material by a :~ 25 predetermined amount. The amount of stretching (e.g. the distance of travel o~ the movable clamp member in a direction away from the opposite end of the graft) is controlled and limited so as not to tear the gra~t and, , . ~. - . ~ .

~ r pre~erai: ly, so ~s not to exceed the degreQ of stretchlng which the graft is capable ~ with~tanding without damage to or weakening of the graftc In c~ses where the graft consists of a segment of artery, the artery segment is pre~erably stretched along its longitudinal axis to a length which is 5% to 60~ and preferably about 5~ to 30% longer than its original unstretched length.
The graft is held in its stretched state (FI&. 2c) lo ~or a predetermined period o~ time and, thereaft~r, the stretching force is removed, thereby allowing the movable clamp member to move back toward the opposite end o~ ~he graft to a point where the graft has returned to a relaxed, unstretched state.
The above-described cycle of stretching-relaxation o~ the collagenous graft material is carried out repeatedly during at least the initial phase or initial time period of the chemical cross-linking reaction.
Exampl~ 2~

Preparatio~ o~ ~ pli~bl~ coronary artary by~a~ raft unda~ d~namic longitu~inal ~t~etch~n~.

This example is illustrated in Figures 2a-2e.
A segment of bovine coronary artery lOb is harvested, cleaned and ligated in the manner described in Example 1. Af~er such harvesting, cleaning and ligating has been completed, the segment of artery lOb is slidably advanced onto the outer surface of a cylindrical upport member 12 or rod. The outer diameter o~ support member !

12 i5 2q[Ual to or slightly larger than the inter~al diameter of the segment of artery lob.
After the se~ment of artery lOb has been disposed upon the support member 12, a ~irst end 13 o~ the artery 5 is ~irmly ~ixed to the support member 12 by a ligature 15. A movable clamp member 18 is attached to the opposite end of th~ artery. Th~ artery is then immersed in a chemical bath 16 containing the 2% (vol.) Denacoltm Ex-313 solution described in Example 1. As in Example 1, lo the solution within the bath is maintained at room temperature.
Immediately after the segment of artery lOb has been immersed in the solution within the bath 16, the end of the art ry to which the movable clamp m mber 18 is attached is pulled away from the opposite end o~ ~he artery (arrow A~ so as to longitudinally stretch the artery to a length which is 20~ longer than its original unstretched length (FIG. 2c).
The amount o~ stretching necessary to achieve the des~red pliability of the fixed graft will vary depending on the types of graft tissue being employed and the particular ~ixant being used. Typically, ~or arterial seqments, the length of the segmant will be increased by 5% to 60% of its original unstretched length. Car~
should be taken to ensur~ that the stretching of the graft does not exceed the elastic capabilities of the yra~t, as such may cause possible perforation, tearing or weakness of the graft. It is desirable that the amount rJ,~

of stretching applied be within the range of that which the graft w~uld encounter during no~mal physiological functioning.
In this ~xample, the segment of artery lOb is maintained in the stretch~d confiyuration ~FIG. 2c) for approximately 2 to 5 seconds. Thereafter, the movable clamp member 1~ is released and moved in the direction of Arrow B t9 a point where the segment of artery lQb has returned to an unstretched, r~laxed state. This lo stretch/release cycle is repea~ed once every 1 to 5 minutes during the initial 0.1 hour to 2 hours of exposure to the 2% Denacol EX-313 fixative (i.e. 30 stretch~relax cycles are carried out during the ~irst hour of exposure to the 2% Denacol Ex-313 fixati~e)~
In this example, during the initial 2 hours o~
fixation, the Denacol Ex-313 solution was replaced 4 time~ (i.e. once every 30 minutes) to ensure optimal reactivity of the solution.
The preferred overall fixation period for thi~ 2%
Denacoltm Ex-313 ~ixative is at least 48 hours~ Thus, a~ter tha repetitive stretch-relax process has been completed during the initial 2 hours oP exposure to the ~ix~tive, the artery lOa was subsequently allowed to remain immersed in the chemical fixative, the movable clamp member 18 may be removed from the free end oP the segment oP artery lob and a lid 17 may be placed on the bath 16 as shown in Figure 2d, so as to ~orm a pressure tlght seal therein. A ~luid pressure sourcs P is then 3 'j 7 applied to the solution within the bath 16 so as to maintain tha ~olution under ~ pressure of approximately 30 mm-Hg. Aftex the entire forty eight (48~ hours F

fixation ~ime period has elapsed, the pressure ~ource P
is removed ~rom the fixative bath 16, the lid 17 i5 removed, the ligature 15 is unti~d and the segment of artery lOb is slidably extracted and remov~d from the cylindrical support member 12~
Thereafter, the segment of artery is washed and stored in a suitable liquid such as ethanol.
ii. Dvnamic Radial Stretchin~
With respect to luminal or tubular graft materials, another means of dynamically, manipulatiny or moving the collagenous graft material during fixation is to subject the collagenous graft material to repetitive radial stretching during the chemical cross-linking process.
In accordance with this aspect of the invention, as illu~trated in Figures 3a-3b, the graft material may be ~ixed on the outer ~urface of a hollow tube, rod, mandrel 2 0 plate, j ig or other support member and the ends of the graft material are ~irmly affixed thereto by way of ligatures, clips, tie wraps or other fixture means.
Thereafter, the graft material is immersed or otherwise exposed to a fixant solution and pressurized fluid is :25 passed into the lumen of the tubular or annular graft, : between the ligaturest ties, clamps or other fixture members, thereby causing the graft to radially dilate or stretch. Thereafter, the fluid pressure within the graft 2 ~ ~3 ~ ~ r t~
_~2W
is decreased, allowing the graft to return to a relaxed u~stretched state.
The amount of radial stretching of the graft is controlled and limited so as not to tear the gra~t and, preferably, so as not to exceed the degx~e o radial stretching which the gr~ft is capable of withstanding without damage to or weakening of the graft.
In cases wherP khe graft consis~s o~ a segment of artery, the artery segment is preferably radially stretched to a radius which is 1~ to 15% larger than the original unstretched radius of the segment of artery.
The graft material is held in the radially stretched state for 3 to 5 seconds and immediately thereafter the pressure within the gra~t is rendered equal to the pressure surrounding the gra~t 50 a to return the graft to its relaxed, unstretched state. The graft is allowed to remain in such relaxed, unstretched state for 2 to 30 minutes and preferably 5 to 10 minutes. Thereafter, this cycle of radial stretching-relaxation, as described herein, is repeated.
The above-described cycle of radial stretch~ng-relaxation is carried out repeatedly during at least the initial portion of the collagen cross-linking reaction.
In instances where slow reacting cross-linking agents : 25 (e.g. polyepoxy compounds) are used it is typically desirable to carry out the dynamic radial stretching-relaxation process throughout the first 1 to 2 hours of exposure to the fixative agent. Thereafter, the dynamic . -23-radial stretching-r~laxation p~c~ss is terminated and the graft may be allowed to remain immersed in ~he slow acting fixant solution at atmospheric or greater th~n atmospheric pr~ssures for an additional period of 24 or more hours until the collagen cross-linking reaction has reached its desired point of compl~tion.
The following Example No. 3 illustrates one application of dynamic radial stretching as a means for preparing pliable coronary artery bypass grafts of lo biological origin.
~empl~ 3:

Prep~ratio~ o~ a pl~abls coronary ar~er~
bypa~ ~r~ft via d~ami~ radial ~tr~tching.

This example is illustrated in Figures 3a-3b.
A segment of bovine coronary artery lOc is harvested, cleaned and ligated as described in Example 1.
After the harvesting, cleaning and ligation of the segment of artery lOc has been completed, the segment of artery lOc is slidably advanced onto the outer surface of a hollow cylindrical support member 12a. The hollow cylindrical support member 12a has a closed distal end 19 and a hollow inner boar extending therethrough. A
plurality o~ fluid outflow apertures 36a are formed in the mid-region of the cylindrical support member 12a so as to reside adjacent the luminal surface of the artery lOc when disposed on the cylindrical support member 12a.
After the segment of artery lOc has been slidably advanced on to the outer surface of the cylindrical -2~-support member 12a ~uch that the ~luid outflow ap~rtures 36a reside adjacent the luminal surface of the artery loc, ligatures 14 are tied about either end of the artery to securely fix the end of th~ artery to the cylindrical support memb~r 12a. It is preferable that ths ligatures 14 be tied sufficiently tight so as to withstand a build up of positive pressure within the segment o~ artery lOc which is up to approximately 5 psi greater than the pressure out~ide of the artery lOc.
lo A first liquid supply tube 21 is in fluid communication with the inner boar of the cylindrical support member 12a and is attached to a container of the 2% Denacoltm ~x-313 fixative solution having a variable pressure apparatus for varying the pressure P1 f fixative solution flowing into the inn~r bore of the cylindrical support member 12a through the liquid supply tube 21 and ultimately within the lumen of the artery lOc. Any resident air or gas is purged from the liquid supply tube 21 and the inner bore of the cylindrical support member 12a such that the liquid supply tube 21 and the entire inner boar of the cylindrical support member 12a are ~illed with the fixative liquid and free o~ air bubbles.
With the segment of artery lOc disposed on the : 25 cylindrical support member 12a, the support member 12a ~: ~ and artery loc are immersed in a bath 16a filled with the : 2% Denacoltm Ex-313 fixative solution. A lid 17a is placed over the bath 16a to form a liquid tight seal .

2 ~

thereon. A second fluid supply tube 23 passes into the closed inner chamber of ~he bath 16a arad is attached to a second container o~ the 2% Denacoltm Ex-313 fixative solution. This second container of ~ixati~e solution is also provided with a variable pressure apparatus ~or varying ~he pressure P2 f fixant liquid passing through the second f luid supply tube 2 3 and into the bath ~6a.
Immediately after the artery lOc has become immersed in the bath 16a and the lid 17a has been firmly mounted in pla~e, the pressure Pl on the first fixative container and the pressure P2 on the second ~ixative container are adjusted, ralative to one another, such that the pressure of fixative liquid in the bath 16a surrounding the artery lOc is approximately 0.1 to 5.0 psi less and preferably 0.1 to 2.5 psi less than the pressure of fixative liquid within the l~men of the artery lOc. As a result, the pressure of fixative liquid within tha artery lOc, relative to the pressure surrounding the artery lOc, will cau~e the artery lOc to radially stretch or dilate as shown in Figure 3a.
Re~erring to Figure 3a, in this example, Pl (stretched) is about 3.0 psi while P2 (stretched) is about 0.5 psi. The resultant pressure differential of 2.5 psi causes the artery lOc to stretch to a stretched radius that is approximately 8% greater than its relaxed unstretched radius. The artery lOc is held in such radially stretched state for 3 to 5 seconds and, therea~ter, the pres~ures Pl trelaxed) and Y2 (relaxed) 2'g~
--2~i--are made e~ual to one another su~h that the segment of artery lOc will return to a relaxed unstrQtched ~tate a~
~hown in Fiyure 3b.
The above-described radial str~t~hing-relaxation s cycle is repeated 30 times during the ~irst hour of exposure to the î ixant solution ( about once every two minutes during ~he first hour)O Thereafter, the pressure P1 on the luminal surface of the artery 1oc is made to equal atmosph~ric pressure and the pressure P2 on the 10 bath solution surxounding the artery lOc is made to equal approximately 3 psi. Such 3 psi pressure P~ is maintaine~ ~or the 47-hour period ~ollowing the ~irst hour of ~ixation during which the radial stretching-relaxation cycle was carried out. At the end of the 48--hour total exposure to the fixant solution with~n bath 16a, th~ pressure P2 is made e~ual to atmos~heric, the lid 17a is removed, ligatures 14 are removed and the segment of artery lOc is slidably extracted from the outer surfacQ o~ the cylindrical support member 12a.
Thereafter, the segment of artery lOc is washed and stored in a Ruitable liquid (e.g. ethanol).
iil. Dynamic Radial Contraction With respect to luminal or tubular graft material~, le.g. segments of blood vessel) another means o~
dyna~ically moving or manipulating the collagen fibers within the gra~t during chemical fixation is to subject the collagenous graft material to repetitive radial contraction during the chemical cross-linking process.

~ r~

In accordance with this aspect of the invention, the gra~t material is disposed on the outer sur~ac~ o~ a hollow tube, rod, mandrel, plate, jig or other generally cylindrical support member having ends which are at least 5 equal in diameter to the inner luminal diameter of the ~
unstretched, r~laxed graft and a mid-region (between the L
two ~nds) which has a diameter less than the inner luminal diameter of the unstretched, relaxed graft, and the ends of the graft material are firmly affixed thereto 10 by way of ligatur~s, clips, tie wraps or other ~ixture means. Thereafter, the graft material is immersed or otherwise exposed to a fixant solution. After immersion in the fixant solution a pressurized ~luid (e~g. more ¦
~ixant solution) is passed onto the exterior wall o~ the 15 tubular or annular graft, between the ligatures, ~ies, clamps or other fixture members, thereby causing the graft to radially contract against the mandrel or rod.
Therea~ter, the ~luid pressure within the gra~t is decreased, allowing the graft tQ return to a relaxed 20 unstretch~d state.
The amount of radial contraction of the gra~t is controlled and limited so as not to tear the graft and so as not to exceed the degree of radial contraction which the graft is capable of withstanding without damage to or : ~ 25 weaX~ning of the graft.
, In cases where the graft consists of a s~gment of artery, the artery segment is preferably radially contractQd to a radius which is 2~ to 15% smaller, and t 28~
preferably 2% to 10% smaller than the original uncontracted radius of the segment of artery.
The graft material i~ held in tha radially contr2cted state ~or 2 to 5 second~. Immediately thereaftPr, the pressure within the graft is rendered equal the pressure surrounding the gr~ft 50 as to return the graft to a relaxed, uncontracted state and i allowed to remain in said un~ontracted state for a ~econd time period, of about 2 to 30 minutes and pre~erably about lo every 2 ~o 10 minutes.
The above-described cycle of radial contraction-relaxation is carried ouk repeatedly durin~7 at least the ini-tial portion o~ the collagen cross-linking reaction.
In instances where 510w reacting cross-linking agents (e.g. polyepoxy compounds) are used it is typically desirable to carry out the dynamic radial contractionr relaxation process throughout the ~irst 1 to 2 hours o~
exposure to the fixative agent. Thereafter, the dynamic radial contraction-relaxation process is terminated and th2 graft may be allowed to remain immersed in the slow acting fixant solution at atmospheric or greater than 2tmospheric pressures for any additional period (e.g. 24 ~ours or more) until the collagen cross-linking reaction has reached its desired point of completion.
The following Example No. 4 illustrates one application of dynamic radial contraction as a mean~ for preparing pliabl~ coronary artery bypass grafts o~
~iological origin.

-29 ~0~3~7 E~ple.4 ~r~pnratio~ o~ a pl~abl~ aoron~r~ ~rt~ry b~p~s~ u~dex ~a~io r~ial oontraqtio~.
This example is illustrated in Figure 4.

A segment of bovine coronary artery lOd is ~arvested, cleaned and ligated as described in Example 1.

After the harvesting, cleaning and ligation of t~le segrnent of artery lOd has been compl~ted, the segment of artery lOd is slidably advanced onto the surface of a hollow, generally cylindrical support member 12b. The hollow, generally cylindrical support member 12b has an open or hollow inner bore extending therethrough and a fluid tight seal or cap formed on the distal and 34 thereof, thereby s~aling and closing the hollow inner bore of the support member 12b at the distal end 34 thereo~.
The ~irst and second end portions 30 of the support member 12b are of a diameter equal to or slightly greater than the unstretched inner diameter of the segment of artery lOd. Tha mid-region 32 o~ the support member 12b has an outer diameter which is less than the inner luminal diameter of thQ relaxed unstretched segment o~
artery 10d. One o~ more fluid flow apertures 36b are rormed in the mid-region 32 of the support member 12b so a~ to provide inflow and outflow of fluid from the inner bore of the support member 12b.
A first liquid supply tube 21 is in ~luid communication with the inner bore of the reduced diameter support member 12b and is attached to a container o~ 2%

: `

Denacoltm Ex-313 fixative ~olution having a variable pressure apparatus for varyirlg the pressure P1 ~
f ixative solution f lowing into the inner bs~xe o~ the reduced diameter support member 12b through the first li~uid supply tube 21. Any resident air or ga~ is purged ~rom the liguid supply tube 21 and the inner bora of th~
reduc~d diameter support member 12b such that the ~irst liquid supply tube 21 and the entire inn~r bore of ~he support member 12b are filled with fixative liquid and free o~ air bubbles.
With the segment of artery lOd disposed on the reduced diameter support member 12b, the support member 12b and artary lOd are immersed in a bath 16a filled with the 2% Denacol m Ex-313 fixative solution. A lid 17a i~
pl~ced over the bath 16a to ~orm a liquid tight seal thereon. A second ~luid supply tube 23 passes into the clos~d inner chamber of the bath 16~ and is attached to a second container o~ the 2% Denacoltm Ex-313 fixative solution. This second container of f ixative solution is also provided with a variable pressure apparatus ~or varying a pressure P2 of fixant li~uid passin~ through the second ~luid supply tube 23 and into the bath 16a.
Immediately after the artery lOd has become immersed in the bath 16a, the lid 17a is firmly mount~d in place, 25 the pressure Pl on the first fixative container and the : pressure P2 on the second fixative container are adjusted relative to one another such that the pressure of ~ixative liquid in the bath 16a surrounding the artery I

2 1 ~

lod is 0.125 to 5.0 psi greater~ and pre~erably 0.1 to 2.5 psi greaker~ than the pr~ssure of the fixativ~ liquid within the lumen of th~ artery lOd. A~ a result, the pressure of fixative liquid within the artery lOd, relative to the pressure surrounding the artery lOd, will cause the artery lOd to radially contract or draw inwardly about the reduced diameter mid-region 32 o~ the reduced diameter support member 12b, as shown in Figure 4.
Re~erring to Figure 4, in this example, Pl (contracted) is about 0~5 psi while P2 (contracted) is about 3.0 psi. The resultant pressure differential of 2.5 psi causes the artery lOd to contract inwardly upon the reduced diameter mid-region 32 of the support member 12b. In this example, the reduced diameter mid-region 32 of the support member 12b is 8~ smaller in diameter than the relaxed un~tretched diameter of the artery lOd, thus this 2.5 psi pressure differential will result in an 8%
decrease in the diameter of the artery lOd. ~he artery lOd is held is such radLally contracted state for 3 to 5 seconds and, thereafter, the pressures P1 (relaxed) and P2 (relaxe~) ar~ made equal to one another ~uch that the segmen~ of artery lOd will return to a relaxed, uncontracted state. ~he segment of artery wa~ then allowed to remain in such relaxed, uncontracted state for a period of about 2 minutes.
The above-described radial contraction-relaxation cycle is r~peated about 30 timQS during the first hour of 5 rl~ I

exposure to the fixant s~lution (about ~nce every 2 minutes during the ~irst hour~. Thereafter, the pressure Pl on a luminal surface of the artery lOd and the pressure P2 within the bath 16a are made equal such that the artery lOd will assum* an un~ontracted, unstretche~
~tate. The artery is then permitted to remain in such uncontracted, unstretch~d state, with fixant solution inside and outside the artery for an additional 47-hour period following the fir~t hour of fixation during which the radial contraction-r laxation cycle was carxied out.
At the end o~ the 48-hour total exposure to the fixant s~lution within bath 16a, the pressure P2 is m~de equal to atmospheric, the lid 18a is removed, ligatures 14 are removed and the segment o~ artery lOd is slidably extracted from the outer surface of the reduced diameter support member 12b.
Thereafter, the segment o~ artery lOd is washed and stored in a suitable liquid (e.g. ethanol).
It will be understood that the invention has been descxibed herein with specific re~erence to certain preferred embodiments and certain specific examples. It will be appreciated that those skilled i~ the art may make numerous additions, modifications, alterations and variations to such preferred embodiments and examples without departing from the spirit an~ scope of the invention. For example, although the in~ention has been described herein with particular reference to arterial vascular gra~ts, it will be appreciated that th~ methods of the present invention may be applied to virtually any collagenous biological tissue including, but not limited ~o, skin grafts, heart valve annulae, heart valve leaflets, conduit grafts and ureter grafts. It will be further appreciated khat specially shaped jigs, ~ixtures or support m~mbers may be devised for holding such tissues during compression andtor stretching thereof in accordance with the present invention. Also, with respect to specific examples disclosed, it will be appreciated that the time course of the treatment and/or exposure times to the fixative reagents may be varied without departing from the methodology of the present invention. For example, it may be desirable to dynamically stretch, compress or contract a segment of blood vessel approximately 72 times a minute so as to mimic the normal heart rate and normal pulsatile movement that would be encountered by the blood vessel in situ.
Accordingly, it is intended that all foreseeable additions, modi~ications, alterations and variations, including but not limited to those specifically mentioned ~erein, not bQ included within the scope of the following clalms.

Claims (52)

WHAT IS CLAIMED IS:

1. A method of preparing a collagenous biological graft material comprising a network of collagen fibers, said method comprising the step of:
(a) altering the relative positioning of the collagen fibers while exposing the collagen fibers to a collagen cross-linking reagent.

2. The method of Claim 1 wherein step (a) comprises:
compressing the network of collagen fibers while exposing the collagen fibers to a collagen cross-linking reagent.

3. The method of Claim 1 wherein step (a) comprises:
alternately stretching and relaxing the network of collagen fibers while exposing the collagen fibers to a collagen cross-linking reagent.

4. The method of Claim 3 wherein step (a) comprises:
alternately compressing and relaxing the network of collagen fibers while exposing the collagen fibers to a collagen cross-linking reagent.

5. The method of Claim 2 wherein the step of "compressing the network of collagen fibers while exposing the collagen fibers to the collagen cross-linking reagent" further comprises:
i. placing the graft material on a smooth surfaced support member;

ii. exerting a compressive force on said graft material to place said graft material in a compressed state, iii, maintaining the graft material in said compressed state;
iv. immersing the compressed graft material in the collagen cross linking reagent for a period of time sufficient to effect adequate fixation of the collagen within the graft material;
v. removing the graft material from the collagen cross-linking reagent; and vi. removing the graft material from said compressed state and from the support member.

6. The method of Claim 5 wherein step (ii.) further comprises:
compressing said graft material to a compressed state whereby the size of the graft material is decreased in at least one dimension by an amount equal to about 1% to 50% of its original uncompressed size in that dimension.

7. The method of Claim 5 wherein step (ii.) further comprises:
compressing said graft material to a compressed state whereby the size of the graft material is decreased in at least one dimension by an amount equal to about 5% to 30% of its original uncompressed size in that dimension.

8. The method of Claim 5 wherein step (iv.) comprises immersing the graft material in a liquid solution of polyepoxy fixative for at least 24 hours.

9. The method of Claim 8 wherein "immersing the graft material in a liquid solution of polyepoxy fixative" comprises:
immersing the graft material in a 2% (vol.) aqueous solution of glycerol triglycidyl ether.

10. The method of Claim 3 wherein the graft has a longitudinal axis and wherein the step of "alternately stretching and relaxing the collagen network while exposing the graft to a collagen cross-linking reagents further comprises:
i. placing the graft material on a smooth surfaced support member;
ii. immersing the graft material in the collagen cross-linking reagent;
iii. stretching the graft material along its longitudinal axis to a longitudinally stretched state;
iv. maintaining the graft material in said longitudinally stretched state for a first period of time;
v. returning said graft material to a relaxed unstretched state;
vi. maintaining the graft material in said relaxed unstretched state for a second period of time;

vii. removing the graft material from the collagen cross-linking reagent; and viii. removing the graft material from said support member.

11. The method of Claim 10 wherein steps (iii.) through (vi.) are carried out repeatedly.

12. The method of Claim 10 wherein steps (iii.) through (vi.) are repeated approximately once every 1 to 5 minutes.

13. The method of Claim 10 wherein steps (iii.) through (vi.) are repeated once every 2 minutes.

14. The method of Claim lo wherein steps (iii.) through (vi.) are repeated every 1 to 5 minutes during the initial 0.1 to 2.0 hours of exposure to the collage cross-linking reagent.

15. The method of Claim 10 wherein said first period of time is approximately 2 to 5 seconds.

16. The method of Claim 10 wherein said second period of time is approximately 1 to 5 minutes.

17. The method of Claim 10 wherein step (ii.) comprises immersing the graft material in a liquid solution of polyepoxy fixative for at least 24 hours and wherein steps (iii.) through (vi.) are repeated approximately once every 1 to 5 minutes during approximately the initial 2 hours of exposure to the polyepoxy fixative solution and, thereafter, the graft is allowed to remain immersed in the polyepoxy fixative solution in its relaxed unstretched state for the remainder of the said at least 24 hour exposure to said polyepoxy fixative.

18. The method of Claim 10 wherein the step of "immersing the graft material in a liquid solution of polyepoxy fixative" comprises:
immersing the graft material in a 2% (vol.) aqueous solution of glycerol triglycidyl ether.

19. The method of Claim lo wherein step (ii.) comprises:
immersing the graft material in a 2% (vol.) solution of glycerol triglycidyl ether for at least 24 hours; and, wherein;
steps (iii.) through (vi.) are repeated every 1 to 2 minutes during at least the first hour of immersion in the glycerol triglycidyl ether solution.

20. The method of Claim 3 wherein said graft material is of a tubular configuration having a generally constant unstretched radius and wherein the step of "alternately stretching and relaxing the network of collagen fibers while exposing the collagen fibers to a collagen cross-linking reagent" further comprises:
i. placing the tubular graft material on the outer surface of a generally cylindrical support member;
ii. immersing the graft material in a collagen cross-linking reagent;

iii. radially stretching the tubular graft material to a radially stretched state;
iv. maintaining the tubular graft material in said radially stretched state for a first period of time;
v. returning the graft material to a relaxed unstretched state;
vi. maintaining the graft material in said relaxed unstretched state for a second period of time;
vii. removing the graft material from the collagen cross-linking reagent;
viii. removing the graft material from the support member.

21. The method of Claim 20 wherein step (ii.) comprises immersing the graft material in a polyepoxy fixative solution for at least 24 hours and wherein steps (iii.) through (vi.) are repeated once every 2 to 30 minutes during the initial 0.1 to 2.0 hours of exposure to the collagen cross-linking reagent.

22. The method of Claim 20 wherein said first time period is 3 to 5 seconds.

23. The method of Claim 20 wherein said second time period is 2 to 30 minutes.

24. The method of Claim 23 wherein said second time period is 5 to 10 minutes.

25. The method of Claim 20 wherein said tubular graft material is stretched to a radius which is 1% to 15% larger than the original unstretched radius of the tubular graft material.

26. The method of Claim 4 wherein the graft material is of a tubular configuration having a generally consistent unstretched radius and wherein the step of "alternately compressing and relaxing the network of collagen fibers while exposing the collagen fibers to a cross-linking reagent" further comprises:
i. placing the tubular graft material on a generally cylindrical support member having a reduced diameter mid-region which is smaller in diameter than the unstretched radius of the tubular graft material;
ii. immersing the graft material in the collagen cross-linking reagent;
iii. exerting a compressive force on said tubular graft material to place the graft material in a radially contracted state about the reduced diameter portion of said support member;
iv. maintaining the tubular graft material in said radially contracted state for a first period of time;
v. returning said tubular graft material to a relaxed uncontracted state;
vi. maintaining the graft material in said relaxed uncontracted state for a second period of time; and vii. removing the graft material from the collagen cross-linking reagent;
viii. removing the graft material from said support member.

27. The method of Claim 26 wherein step (ii.) comprises immersing the graft material in a polyepoxy fixative solution for at least 24 hours and wherein steps (iii.) through (vi.) are repeated once every 2 to 30 minutes during the initial 0.1 to 2.0 hours of exposure to the collagen cross-linking reagent.

28. The method of Claim 26 wherein said first time period is 2 to 5 seconds.

29. The method of Claim 26 wherein said second time period is 2 to 30 minutes.

30. The method of Claim 29 wherein said second time period is 2 to 10 minutes.

31. The method of Claim 26 wherein said tubular graft material is contracted to a radius which is 2% to 15% smaller than the original uncontracted radius of the tubular graft material.

32. The method of Claim 26 wherein said tubular graft material is contracted to a radius which is 2% to 10% smaller than the original uncontracted radius of the tubular graft material.

33. A biological vascular graft prepared by a method which comprises the steps of:
(a) harvesting a segment of blood vessel from a mammalian source;

(b) placing the segment of blood vessel on the outer surface of a cylindrical support member such that the luminal surface of the blood vessel is in contact with the outer surface of the cylindrical support member;
(c) longitudinally compressing the segment of blood vessel to a compressed length whereby the segment of blood vessel is shortened by an amount equal to 1% to 50% of its original relaxed length;
(d) immersing the longitudinally compressed segment of blood vessel in the collagen cross-linking reagent for a period of time sufficient to achieve adequate cross-linking of the collagen contained in the segment of blood vessel;
(2) removing the segment of blood vessel from the collagen cross-linking reagent;
(f) releasing the segment of blood vessel from said longitudinally compressed state and removing the segment of blood vessel from the outer surface of said cylindrical support member.

34. The biological vascular graft of Claim 33 wherein step (c) comprises:
longitudinally compressing the segment of blood vessel to a compressed length whereby the segment of blood vessel is shortened by an amount equal to 40%
to 50% of its original relaxed length.

35. A biological vascular graft prepared by the method comprising the steps of:

(a) harvesting a segment of blood vessel from a mammalian source;
(b) immersing the segment of blood vessel in a collagen cross-linking reagent;
(c) exerting a longitudinal stretching force on the segment of blood vessel to stretch the segment of blood vessel to a stretched length;
(d) holding the segment of blood vessel in said stretched length for a first period of time;
(e) removing the longitudinal stretching force and allowing the blood vessel to return to an unstretched relaxed state;
(f) maintaining the blood vessel in said unstretched relaxed state for a second period of time;
(g) repeating steps (d) through (g) every 1 to 5 minutes during the initial 0.1 to 2.0 hours of immersion in the collagen cross-linking reagent;
(h) removing the segment of blood vessel from the collagen cross-linking reagent;
(i) removing the segment of blood vessel from the cylindrical support member.

36. The biological vascular graft of Claim 35 wherein:
step (c) of the preparation method further comprises:
immersing the segment of blood vessel in a liquid solution of polyepoxy fixative for a period of at least 24 hours; and step (g) of the preparation method further comprises:
repeating steps (d) and (e) approximately once every 2 minutes during at least the initial 1 hour of immersion in the polyepoxy cross-linking reagent.

37. The biological vascular graft of Claim 35 wherein the method of preparation further comprises the steps of:
placing the segment of blood vessel on the outer surface of a cylindrical support member such that the luminal surface of the blood vessel is in contact with the outer surface of the cylindrical support member; and, after the blood vessel has been removed from the collagen cross-linking reagent, removing the segment of blood vessel from the cylindrical support member.

38. A biological vascular graft prepared by the method comprising the steps of:
(a) harvesting a segment of blood vessel from a mammalian source;
(b) immersing the segment of blood vessel in a collagen cross linking reagent;
(c) exerting a radial stretching force on the segment of blood vessel to radially stretch the blood vessel to a radially stretched state;

(d) maintaining the blood vessel in said radially stretched state for a first period of time;
(e) removing the radial stretching force and allowing the blood vessel to return to a relaxed unstretched state;
(f) maintaining the blood vessel in said relaxed unstretched state for a second period of time;
(g) repeating steps (c) through (f) approximately once every 2 to 30 minutes during the initial 0.1 to 2.0 hours of exposure to the collagen cross-linking reagent;
(h) removing the segment of blood vessel from the collagen cross-linking reagent.

39. The biological vascular graft of Claim 38 wherein:
step (b) of the preparation method comprises immersing the segment of blood vessel in a liquid polyepoxy fixative solution for a period of at least 24 hours; and step (f) of the preparation method comprises repeating steps (c) through (f) approximately once every 2 minutes during at least the first hour of immersion in the polyepoxy fixative solution.

40. The biological vascular graft of Claim 38 wherein the first time period is 3 to 5 seconds.

41. The biological vascular graft of Claim 38 wherein the second time period is 2 to 30 minutes.

42. The biological vascular graft of Claim 38 wherein step (c) comprises exerting a radial stretching force on the segment of blood vessel to radially stretch the blood vessel to A radially stretched state wherein the radius of the blood vessel is approximately 1% to 10%
larger than its original unstretched radius.

43. The biological vascular graft of Claim 38 wherein step (c) comprises exerting a radial stretching force on the segment of blood vessel to radially stretch the blood vessel to a radially stretched state wherein the radius of the blood vessel is approximately 2% to 10%
larger than its original unstretched radius.

44. The biological vascular graft of Claim 38 wherein step (c) comprises exerting a radial stretching force on the segment of blood vessel to radially stretch the blood vessel to a radially stretched state wherein the radius of the blood vessel is approximately 8% larger than its original unstretched radius.

45. The biological vascular graft prepared by the method comprising the steps of:
(a) harvesting a segment of blood vessel from a mammalian source;
(b) immersing the segment of blood vessel in a collagen cross-linking reagent;
(c) exerting a compressive force on the segment of blood vessel to radially contract the blood vessel to a radially contracted state wherein the radius of the blood vessel is approximately 2% to 15% smaller than the original uncontracted radius of the blood vessel;
(d) holding the blood vessel in said radially contracted state for a first period of time;
(e) removing the compressing force and allowing the blood vessel to return to a relaxed uncontracted state;
(f) allowing the blood vessel to remain in said relaxed uncontracted state for a second period of time;
(g) repeating steps (c) through (f) every 2 to 30 minutes during the initial 0.1 to 2.0 hours of exposure to the collagen cross-linking reagent;
(h) removing the segment of blood vessel from the collagen cross-linking reagent.

46. The biological vascular graft of Claim 45 wherein:
step (b) comprises immersing the segment of blood vessel in a liquid polyepoxy fixative solution for at least 24 hours; and step (g) comprises repeating steps (c) through (f) approximately once every 2 minutes during at least the first hour of immersion in the polyepoxy fixative solution.

47. The biological vascular graft of Claim 45 wherein the first time period is 2 to 5 seconds.

48. The biological vascular graft of Claim 45 wherein the second time period is 2 to 30 minutes.

49. The biological vascular graft of Claim 48 wherein the second time period is 2 to 10 minutes.

50. The biological vascular graft of Claim 45 wherein the preparation method further comprises the steps of.
affixing the segment of blood vessel to a reduced diameter cylindrical member having a diameter which is smaller than the relaxed uncontracted diameter of the segment of blood vessel; and wherein step (c) of the preparation method further comprises:
exerting a compressive force against the blood vessel to cause the blood vessel to assume a contracted state wherein the blood vessel is reduced in diameter and the luminal surface of the blood vessel is pressed in annular contact with the outer surface of the reduced diameter cylindrical support member;
and wherein the preparation method further comprises the final step of (h) removing the segment of blood vessel from the reduced diameter cylindrical support member.

51. A method of improving the pliability of collagenous biological graft materials, said method comprising the step of:

altering the locations of the chemical cross-linkages formed during the collagen cross-linking process.

52. A method of improving the pliability of chemically cross-linked collagenous biological graft materials, said method comprising the step of:
altering the orientations of chemical cross-linkages formed during the collagen cross-linking process.